1. Field of the Invention
The imbalance in the ecosystem, human health hazards and the development of resistance by some insect pests caused by the continuous use of conventional insecticides have necessitated the search for alternative safer pesticides. Botanicals by virtue of their relatively low toxicity, biodegradability and many other factors acceptable to the environment are considered as the best alternatives to toxic chemicals. Among Botanicals, neem tree ranks first in view of its excellent pest control properties, its low mammalian toxicity and relative abundance in countries like India, Pakistan, Burma, Sri Lanka and Africa. No other tree or plant possessing insect-control properties, has received as much attention as neem world over which is evident from the three International and one National (India) conferences held during the past decade.
2. Description of the Prior Art
Although the protection of stored food grains, woolen clothes, fruits and vegetables by neem leaves has been used in India since time immemorial, the first report of the pesticidal properties appeared around 1927 when Mann and Burns Agri. J. India, Calcutta 22, 325 (1927)! observed during the locust cycle of 1926-27 that adult locusts did not feed on neem leaves. This was followed by Chopra Rept. of Dept. of Agri., Punjab Pt.2, Vol. 1 P.67 (1928)! who treated the extract of neem leaves as contact poison on grub weevils. Since then a number of publications describing the various activities from different parts of neem tree have appeared. For example, neem oil has shown antifeedant activity against Nephotettix virescens (leaf hopper of rice). Neem Newsletter 1 (3) 28 (1984)!. Neem seed extract has shown antifeedant activity against Mythimna separata (rice ear cutting caterpillar) Neem Newsletter 1 (3), 31 (1984)!. Neem cake has exhibited antifeedant activity against Nilaparvata lugens (Rice Brown Plant Hopper) J. Economic Entol., 77, 502 (1984)!. Neem oil has shown oviposition deterrent and ovicidal activity against a stored grain pest of rice Corcyra cephalonica (rice moth). Neem seed kernel water suspension has shown oviposition deterrent activity against Spodoptera litura (Tobacco caterpillar) Phytoparasitica 7(3), 199 (1979)!. Neem leaves extract has shown antifeedant activity against Rhizopertha dominica (lesser grain borer) Neem Newsletter, 1 (2), 20 (1984)!. Neem root exudates have been reported to contain allelochemicals.
The first active principle exhibiting antifeedant properties against locusts (Schistocerca gregaria) was isolated in 1968 which was named as azadirachtin by Morgan and Butterwoth Chem.Commn.23 (1968)!. Since then azadirachtin has been shown to exhibit insect antifeedant and/or growth-inhibitory activities on more than 50 insect species. For example it has exhibited antifeedant activity at a dosage of 0.005% on Earias fabia (spotted cotton ballworm, family, Lepidoptera) Phytoparasitica, 9 (1), 27 (1981)!. It has shown growth inhibitory activity at a dosage of 5-10 ppm against Spodoptera litura (tobacco caterpillar, Lepidoptera Proceedings of the National Seminar on Neem in Agri.,! IARI, New Delhi, (1983); Indian J Expt. Biol. 23(3), 16 (1985) and LD.sub.50 of azadirachtin against S. litura is reported to be 1.1 g/g J. Entomol. Res., 11 (2), 166 (1987)!. It has been reported to exhibit sterilant, insecticidal, delay in egg hatching, growth inhibitory and antifeedant activities at a dosage of 10-100 ppm against Epilachna verivestis (Maxican bean beetle, Coleoptera Z pflakrankh pflaschutz, 2 (3), 176 (1975); Z. Angew Entomol, 93, 12 (1982);
Systemic action of azadirachtin through roots to leaves has been reported by Saxena et al J. Econ. Entomol., 77 (2) 502 (1984)!.
The drawing accompanying this specification shows the structure of azadirachtin; in formula 1 which was established in 1986 by three groups of workers Tetrahedron, 43, 2789; 2805, 2817 (1987)!.
Till this time more than 80 tetranortriterpenes and derivatives of azadiraachtin have been isolated from neem extracts. Some of them are mentioned here:--nimbinene, nimbandiol, azadiradione, salannin, vilasinin, gedunin, meldenindiol and nimbin. Out of them at least 35 triterpene derivatives show either insect antifeedant activity growth-inhibitory activity or both. For example azadirachtin A, B, D, E, F and G show insect growth inhibitory activities against Epilachna verivestis at a dosage of 1-10 ppm Insecticides of Plant Origin, ACS Symposium series, 387, 150 (1989)!. Similarly 3-tigloylazadirachtol has exhibited 97% antifeedant activity at a dosage of 1.0 ppm Tetrahedron, 45, 5175 (1989)!. Amongst other compounds azadiradione, azadirone, 6-deacetylnimbinene, epoxyazadiradione, gedunin, nimbandiol, salannin and salannol have shown antifeedant and growth-inhibitory activity against E. Verivestis Proceedings 2nd International Neem Conference 181 (1983)!. Azadirachtin has been reported to be active against nematodes and whitegurbs Entomol. Exp. Appl., 24, 448 (1978).
Synthetic insecticides belonging to the class of organochlorine and organophosphorous are found to be quite toxic to mammals, fish, honeybees etc. They pollute the ecosystem by their toxicity and residual action. In addition they cause serious health hazards to human beings. In view of this there is an urgent need to look for safer alternatives and plant products appear to be the right choice because of their relatively low toxicity and biodegradability.
With the above objective we have made extensive research on the pest control properties of the extract from neem seeds and have observed that if the undesirable components viz. the saturated fats and the water soluble compounds including sugars are removed, the resulting extract is enriched with the required active compounds viz. the triterpenoids and the unsaturated fats.
Neem seed extract contains essentially four components namely saturated and unsaturated fats, triterpenoids and water soluble compounds containing sugars. Of these, triterpenoids exhibit insect-antifeedant and growth-inhibitory activity while the unsaturated fats possess insecticidal activity against aphids, mites, jassids, cotton white flies and other soft bodied insects. Of the remaining two, saturated fats are inactive and phytotoxic (above 2% dosage) and the water soluble sugars act as insect attractants and fungal growth promoters. Both these are undesirable in the formulation of pest control agents from neem.
In the known processes of preparing insecticidal composition from neem no attempt has been made to separate all the above said four components so that the concentration of the required compounds, the triterpenoids and the unsaturated fats can be used advantageously for pest control purposes. Some of the processes involve expelling neem oil which contains a low percentage (0.05) of active triterpenoids and directly use for pest control purposes. In some other processes the neem is extracted using a solvent like (hexane) to obtain an oil containing low percentage (0.05) of the active triterpenoids. Consequently, due to the very low percentage of the terpenoids these oils used directly for pest control purposes do not have desirable pest control properties.
A mixture of neem oil, karanja oil, mohua oil, gingely oil, castor oil has also been used but it exhibits phytotoxic properties in field trials. Neem based fertilizer in which neem extractive (crude) is blended with urea and sold as a fertilizer is also commercially available.
In the process of the present invention we have removed all the drawbacks of the existing processes by complete extraction of neem seed powder and solvent partitioning in one step using two immiscible solvents, forming two layers, one solvent layer containing the lipids and the other solvent layer containing the triterpenes and water soluble compounds including sugars. The second solvent layer is treated with a polar solvent in which the triterpenes only are soluble, thus separating them from the undersired sugar fraction by filtration/decantation. Thus, in two steps the active rich triterpenoid fraction exhibiting insect antifeedant and growth-inhibitory activity is obtained which is free from the undesirable lipids and water soluble compounds including sugars. The percentage of the active triterpenoid fraction is about 2.5% based on the seed powder.
Similarly the separation of the undesirable saturated fat (9%) from the total lipids has been achieved by fractional crystallisation from acetone at 0-10.degree. C. The saturated fats, at the above temperature solidify and are removed by filtration and the unsaturated fats remain in the mother liquor.
During our continued research in the field of pest control agents we observed that, 1) neem seed extract is more active biologically than the extract of any other part of the neem tree. 2) Neem seed extract is more active than neem seed kernel extract as the husk or hull of the seeds also contains active principles. 3) Neem seed extract is more active than the expelled oil. 4) Triterpene fraction of the seed extract exhibits antifeedant activity and insect-growth inhibiting activity, while the lipid fraction rich in unsaturated fats is responsible for aphidicidal, miticidal and insecticidal activity against sucking pests and soft bodied insects.